1. Field of the Invention
This invention describes methods to prepare specific combinations of metallo-lactoferrin (LF)-coenzyme mixtures to trigger the release of bioenergy (bio-E) in the form of adenosine triphosphate (ATP). Additionally the invention discloses compositions of functional delivery systems to recreate physiological proton gradients for rapid activation and release of cellular and extracellular ATP.
2. Description of the Related Art
All living organisms, plants and animals, operate a power house of bio-E for physiological functions. The bio-E is required for metabolic processes that keep organisms alive. Some of these processes occur in a continuous manner, such as the metabolism of nutrients, synthesis of essential biological molecules (i.e. proteins and DNA), active transport of molecules and ions in/out of the organism. Other processes occur only at specific times, such as a muscle contraction or other cellular movements. Animals obtain their energy by chemo-oxidation of nutrients in the mitochondria, where as plants do so by photo-oxidation, trapping the sunlight using chlorophyll. However, this trapped energy is useful only when it is further transformed into a bio-compatible form that the organism could easily utilize. This specific energy transformer is essentially a nucleotide, the adenosine triphosphate or ATP.
ATP, the bio-E currency or unit, transfers energy from chemical bonds to endergonic (energy absorbing) reactions within the cell. Structurally, ATP consists of the adenine nucleotide (ribose sugar, adenine base, and phosphate group, PO4−2) plus two other phosphate groups. Energy is stored in the covalent bonds between phosphates, with the highest amount of energy (˜7 kcal/mole) in the bond between the second and third phosphate groups. This covalent bond is known as the pyrophosphate bond. Following is the chemical equation for ATP formation: ADP+Pi+ΔE→ATP. The chemical equation for expenditure/release of ATP energy is: ATP→ADP+ΔE+Pi. Thus, the function of ATP to release energy is dependent on losing the endmost phosphate group (covalent bond) by hydrolysis. This enzymatic reaction with ATP releases the bio-compatible energy for cellular processes. The metabolic end product of this process is adenosine diphosphate or ADP, and the phosphate group either ends up as an orthophosphate (HPO4) or attached to another molecule (such as an alcohol). Even more bio-E can be extracted from ATP by dissociating the second phosphate group to produce adenosine monophosphate or AMP. Energy is not immediately needed when an organism is resting. Accordingly, the reverse reaction takes place and the phosphate group is reattached to the ADP using energy obtained from chemo- or photo-oxidation. Therefore, the ATP molecule acts as a chemical transformer, storing energy when it is not needed, but capable to release the bio-E instantly when required by the organism.
The enzyme that makes ATP is the ATPase or ATP synthase, which is present in the mitochondria of animal cells or chloroplasts in plant cells. The energy requiring step in making ATP is not the synthesis from ADP and phosphate, but the initial binding of the ADP and the phosphate to the enzyme. The ATPase enzyme promotes ion transport through membranes and the phosphate group that is ripped from ATP binds directly to the enzyme. Two processes convert ADP into ATP: 1) substrate-level phosphorylation that occurs in the cytoplasm when ATPase attaches a third phosphate to the ADP; and 2) chemiosmosis, which is comprised of several enzymes arranged in an electron transport chain (ETC) embedded in a membrane. In eukaryotes this membrane is either in the chloroplast or mitochondrion. During chemiosmosis, H+ ions (protons) are pumped across the membrane into a confined space that contains numerous hydrogen ions. The energy for pumping comes from the coupled oxidation-reduction reactions in the ETC. Electrons are passed from one membrane-bound enzyme to another, losing some energy with each transfer (as per the second law of thermodynamics). This lost energy allows the pumping of protons against the concentration gradient (there are fewer protons outside than inside of the confined space). The confined protons are restricted to pass back through the membrane. Therefore, their only exit is through the enzyme ATPase, which is located in the confining membrane. As the proton passes through the ATPase, energy from the enzyme is transferred to attach a third phosphate to ADP, converting it to ATP.
Generation of bio-E is dependent on three important factors: 1) the elemental complex (i.e. metal ions Fe3+, Cu2+, Cr2+, Zn2+ and Mg2+) for biosynthesis and function of coenzymes; 2) the coenzyme complex (i.e. coenzyme Q10, nicotinamide adenosine dinucleotide or NADH, Flavones, B-complex vitamins) for transport of charged electrons during oxidative phosphorylation; and 3) the trigger complex (i.e. ATPase and a proton gradient equipped with scavenging and protection against free radicals).
Among the elemental complex, iron is the critical component for the bio-E pathways including the coupling of inorganic phosphate to ADP to form ATP in living organisms. Iron is the most abundant transition metal in mammals and humans and exists in two oxidative states in aqueous solutions, ferrous (Fe2+) and ferric (Fe3+), which allows this metal ion to participate in a broad range of chemical reactions from +350 mV to −500 mV. Such intracellular reactions include the oxidative catalysis of oxygen and hydrogen peroxide, the decomposition of peroxide and superoxide, and oxidative phosphorylation. In the respiratory, photosynthetic and microsomal ETC, iron exists in cytochromes (of the types a, b, c and d) as well as iron-sulfur proteins. Other iron-sulfur proteins catalyze oxidation reactions (xanthine oxidase, xanthine dehydrogenase, aldehyde oxidase and sulfite oxidase) and the Krebs cycle enzyme aconitase. Iron is a critical co-factor for the enzyme RNA reductase during DNA synthesis. Iron reduces the nucleotides ADP, uridine diphosphate (UDP), cytosine diphosphate (CDP) and guanine diphosphate (GDP), forming precursors for the DNA (Jacobs A, Worwood M (ed). Iron in biochemistry and Medicine. Academic Press, NY, pp 529-572, 1980; Crichton R R, Charloteaux-Wauters M. Iron transport and storage. Eur J Biochem 164:485-506, 1987).
Copper is the second important component of the elemental complex, which acts as a catalytic agent via many copper metalloenzymes which act as oxidases. Amine oxidases are important in a variety of physiological processes. Ferroxidases, copper enzymes in the plasma, are required for ferrous iron oxidation and binding of iron to transferrin. The main copper protein in plasma, ferroxidase I (or ceruloplasmin), is a potent antioxidant. Another copper enzyme, cytochrome c oxidase, is a mitochondrial enzyme that catalyzes the reduction of oxygen to water to fuel ATP synthesis. Cytochrome c oxidase is most abundant in highly metabolic tissues, including the heart, brain, and liver. Other copper enzymes are responsible for precursors of dopa and melatonin formation, conversion of dopamine to norepinephrine, production of amides, and protection from free radical damage.
Another component of the elemental complex, zinc (Zn2+) is a catalyst for more than 300 enzymes as well as a cofactor for DNA, RNA, and protein synthesis. Manganese (Mn2+) is required for several metabolic pathways involved in amino acid, cholesterol, and carbohydrate metabolism. Manganese metalloenzymes include arginase, phosphoenolpyruvate decarboxylase, glutamine synthetase, and manganese superoxide. Chromium (Cr2+) as referred to as glucose tolerance factor (GTF) potentiates glucose uptake by cells, oxidation of glucose, and incorporation of glucose into fatty acids and cholesterol. Finally, magnesium (Mg2+) is required in the formation of cyclic AMP (cAMP) and for ionic movements across cell membranes. It is also involved in protein synthesis and carbohydrate metabolism. (Shils M, Olson A, Shike M. Modern nutrition in health and disease. 8th ed. Philadelphia, Pa.: Lea and Febiger, 1994; Covington T R, et al., Handbook of nonprescription drugs, Washington D.C.: Am Pharmaceutical Assn, 1996; 945, 272; Vincent J B. The biochemistry of chromium. J Nutr 130:715-718, 2000).
Among the coenzyme/vitamin-B complex, coenzyme Q is an essential part of the cellular machinery to produce ATP and provides bio-E for vital cellular functions. The major part of ATP production occurs in the inner membrane of mitochondria, where coenzyme Q is found. Coenzyme Q has a unique function since it transfers electrons from the primary substrates to the oxidase system at the same time that it transfers protons to the outside of the mitochondrial membrane. This transfer results in a proton gradient across the membrane. As the protons return to the interior through the enzymatic machinery for making ATP, they drive the formation of ATP. Coenzyme Q is bound to the oriented enzymatic protein complexes. It is oxidized and releases protons to the outside and picks up electrons and protons on the inside of the mitochondrial membrane. There are two protein complexes in the membrane where electrons and protons are transferred through coenzyme Q. The first is the primary reductase where coenzyme Q is reduced by NADH (complex I). During the reduction process four protons are transported across the membrane for every coenzyme Q reduced. It has been suggested that coenzyme Q is reduced and reoxidized in the complex twice before electrons are transferred to a second loosely bound coenzyme Q to form quinol which can travel through the lipid in the membrane to a second complex where the quinol is oxidized again (complex III) with transfer of protons across the membrane. The details of quinol binding and oxidation at the binding site in this complex are well known. As in complex I, there is a cyclic oxidation-reduction-reoxidation with the oxidation and proton release step always on the outside so that protons are released in the right direction. Again the oxidation-reduction cycle allows for four protons to cross the membrane for each quinol oxidation cycle. The quinone cycle thus doubles the efficiency of the coenzyme Q in building up the proton charge across the membrane which allows twice as much ATP production than a simple one step oxidation of quinol. After the cycle is completed the oxidized quinone migrates through the membrane to be re-reduced at complex I. A simpler form of energy conversion based on coenzyme Q reduction-oxidation is found in lysosomes. In this case the quinol transfers a proton across the lysosomal membrane to acidify the inside which involves energy input to work against a proton gradient. No ATP can be formed since the lysosomal membrane does not have a proton driven ATP synthetase. The acidification of the lysosome activates hydrolytic enzymes for digestion of cellular debris. In other words, coenzyme Q energizes cell house cleaning. Details of the enzymes and possible coenzyme Q binding sites in the lysosomal membrane are not known. The enzyme complex in the membrane involves reduction of coenzyme Q by NADH in the cytoplasm and reoxidation of the quinol by oxygen (Brandt U: Proton translocation in the respiratory chain involving ubiquinone—a hypothetical semiquinone switch mechanism for complex I. Biofactors 9: 95-102, 1999; Yu C A, Zhang K-P, Deng H, Xia D, Klm H, Deisenhofer J, Yu L: Structure and reaction mechanisms of the multifunctional mitochondrial cytochrome bc1 complex. Biofactors 9: 103-110, 1999; Gille L, Nohl H. The existence of a lysosomal redox chain and the role of ubiquinone. Arch Biochem Biophys 375: 347-354, 2000).
Coenzyme 1, or NADH, is the active coenzyme form of vitamin B3. NADH is a natural substance found in most life forms and is necessary for energy production. NADH provides input to the respiratory chain from the NAD-linked dehydrogenases of the citric acid cycle. The complex couples the oxidation of NADH and the reduction of coenzyme Q, to the generation of a proton gradient which is then used for ATP synthesis. NADH is located both in the mitochondria and cytosol of cells. It is a dinucleotide comprised of the nucleotide adenylic acid and a second nucleotide in which nicotinamide, a B vitamin, is the nitrogenous base. NADH is a key member of the ETC in mitochondria. The nicotinamide moiety is the portion of the dinucleotide that undergoes reversible reduction. NADH is the reduced form of the dinucleotide. The passage of electrons along the ETC is coupled to the formation of ATP by the oxidative phosphorylation process. The mitochondrial membrane is impermeable to NADH, and this permeability barrier effectively separates cytoplasmic NADH from the mitochondrial NADH pools. However, cytoplasmic NADH can be used for bio-E production. This occurs when the malate-aspartate shuttle introduces reducing equivalents from NADH in the cytosol to the ETC of the mitochondria.
Biotin (coenzyme R) is mitochondrial reserve that acts as a coenzyme in bicarbonate-dependent carboxylation reactions. Biotin-containing enzymes are involved in gluconeogenesis, fatty acid synthesis, propionate metabolism, and the catabolism of leucine. Pyruvate decarboxylase is a biotin-dependent enzyme (Bonjour J P. Biotin in human nutrition. Ann NY Acad Sci 447:97-104, 1985).
Among the B-complex vitamins, thiamine (vitamin B1) is required for carbohydrate metabolism. It combines with ATP to form thiamine diphosphate, a coenzyme in carbohydrate metabolism that facilitates the decarboxylation of pyruvic acid and a-ketoglutaric acid. This coenzyme is also a part of transketolation reactions. Thiamine is also a coenzyme in the utilization of pentose in the hexose monophosphate shunt.
Riboflavin (vitamin B2) is required for tissue respiration. It is converted to the coenzyme riboflavin 5-phosphate (flavin mononucleotide, FMN) and then to the coenzyme flavin adenine dinucleotide (FAD). These act as hydrogen carriers for several enzymes known as flavoproteins, which are involved in oxidation-reduction reactions of organic substrates and in intermediary metabolism. Riboflavin is a cofactor for various respiratory enzymes such as glutaryl coenzyme A dehydrogenase, erythrocyte glutathione reductase, sarcosine dehydrogenase, electron transferring flavoprotein (ETF) dehydrogenase, and NADH dehydrogenase.
Niacin-Niacinamide (vitamin B3) includes niacin (nicotinic acid) and niacinamide (nicotinamide). The term niacin refers specifically to nicotinic acid, but is also used collectively to refer to both nicotinic acid and nicotinamide. Niacinamide is required for lipid metabolism, tissue respiration, and glycogenolysis. Niacinamide is incorporated into the coenzymes, NAD and NADP that act as hydrogen-carrier molecules.
Pantothenic acid (vitamin B5) is required for intermediary metabolism of carbohydrates, proteins and lipids. It is a precursor of coenzyme A, which is required for acetylation reactions in gluconeogenesis, in the release of bio-E from carbohydrates, the synthesis and degradation of fatty acids, and the synthesis of sterols, steroid hormones, porphyrins, acetylcholine and other compounds. Pantothenic acid also appears to be essential to normal epithelial function.
Pyridoxine (vitamin B6) is required for amino acid metabolism. It is also involved in carbohydrate and lipid metabolism. In the body, pyridoxine is converted to coenzymes pyridoxal phosphate and pyridoxamine phosphate, in a wide variety of metabolic reactions. These reactions include transamination of amino acids, conversion of tryptophan to niacin, synthesis of gamma-aminobutyric acid (GABA) in the central nervous system, metabolism of serotonin, norepinephrine and dopamine, metabolism of polyunsaturated fatty acids and phospholipids, and the synthesis of heme, a hemoglobin constituent. Pyridoxine is involved with several of the reactions important for the overall metabolism of nitrogen; therefore, pyridoxine requirements are related to the total amino acid nitrogen burden to be metabolized. Pyridoxine is also a cofactor for enzymes involved in one of two pathways for the metabolism of homocysteine.
Folic acid (vitamin B9) converts to tetrahydrofolate after physiological absorption. In humans, tetrahydrofolate-based coenzymes play a major role in intracellular metabolism and in the rate-limiting steps of DNA synthesis. Folic acid is also involved in the metabolism of homocysteine.
Cobalamin (vitamin B12) is required for nucleoprotein and myelin synthesis, cell growth reproduction, and erythropoiesis. Synthetic vitamin B12 (cyanocobalamin and methylcobalamin) converts to coenzyme B12, which is essential for the conversion of methylmalonate to succinate, and the synthesis of methionine from homocysteine. Vitamin B12 is involved in maintaining sulfhydryl groups in the reduced form required by enzymes involved in fat and carbohydrate metabolism and protein synthesis.